High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication

Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.

pneumonia in patients who are being mechanically ventilated. More notoriously, this organism is also the main pathogen in cystic fibrosis patients and contribute to the chronic colonization of the lungs, leading to destruction and finally their death (1). In fact, P. aeruginosa infection rarely occurs in healthy individuals due to efficient clearance of the pathogen by the innate immune responses that include neutrophils and macrophages (2). This organism is motile via a single polar flagellum.
This whip like appendage is required for swimming motility, chemotaxis, spreading in the environment, ability to translocate to preferred host cells, access to optimal colonization sites, and the Toll-like receptor 5 (TLR5)-dependent inflammatory response (3). TLR5 as a receptor has an important role in adaptive immune responses that recognizes specific pathogen-associated molecular patterns (PAMPs), which are conserved domains unique to microorganisms. By this mechanism, the flagellin activates macrophages, dendritic cells, and airway and corneal epithelial cells to produce proinflammatory mediators (4). The role of flagellum, as a virulence factor, in contributing to bacterial pathogenicity of many microorganisms, including P. aeruginosa, has been well established (5). PAO1 was predicted to be important for binding to TLR5, leading to inflammatory responses (6).
Domains D2 and D3 cover the hypervariable regions exposed as a folded β-sheet structure (7).
On the other hand, the amino acid sequence alignment demonstrates that flagellin proteins are highly conserved in the N-and C-terminal regions, which correspond to D0 and D1 domains, and the variable regions form the outside surface-exposed domains (D2 and D3) in the assembled filament.
Analysis of the crystal structure of P. aeruginosa flagellin suggests that D2 domain has a unique structure of two β-sheets and one αhelix that has not been found in other flagellins. It also has been suggested that the D2 domain would be exposed to solution and could play a key role in immunogenicity (8). aeruginosa (9). It is presumed that the flagellin protein is an effective immunologic factor for immunity in Pseudomonas infections. The main objective of the present study was to express and purify the recombinant type B-flagellin (r-Bflagellin) from P. aeruginosa using a new purification procedure and evaluate its biological activity.

Bacterial strains, and plasmids
Escherichia coli (E. coli) strains BL21 (DE3) and Top10F, as expression and preservation hosts, were preserved in our laboratory. The widely studied strains of P. aeruginosa PAO1 and PAK were used as type B and type A flagellated strains.

Construction of the expression vector
The whole type B-flagellin gene (fliC) was inserted into the E. coli expression vector pET28a, in frame with a T7 promoter, kanamycin resistant gene and the C-terminal six-His-tagged sequence.
The fliC gene containing a BamHI site located at the 5′ end, and a HindIII site located at the 3′ end.

Expression and isolation of inclusion bodies
To over-express the protein, pET28a/fliC construct was transformed into BL21 (DE3) and

Motility inhibition assay
To  in different groups, and the results were compared together.

Statistical analysis
For the statistical analysis, SPSS 18.0 software was used. All data of this study were expressed as mean ± SD. The data were analyzed using one-way analysis of variance (ANOVA) and Student's t-test (Statview). The p-values less than 0.05 were considered to be statistically significant.   and induced sample (Fig.3, lanes 2 and 3). A weak band was observed when the non-induced sample was probed with the antibody (Fig.3, lane 1).

PCR amplification and construction
Overall, our results indicated that the rabbitproduced antibodies are highly specific to detect the r-B-flagellin.

Motility inhibition assay
The biological activity of the immunized rabbit sera was tested in the motility inhibition assay for their functional activity to inhibit the motility of P. aeruginosa strains PAO1 and PAK strains. In this assay, non-immune rabbit serum (NRS) as non-immunized serum and PBS were used as controls. As shown in Table 1 and   The mean diameters of bacterial spreading was measured according to millimeter (mean ± SD). & P < 0.014; *** P < 0.003

Opsonophagocytic killing activity
To In recent years, high-throughput proteinrefolding methods have been developed for the renaturation of inclusion bodies (12,13). These include three methods such as dilution, dialysis or solid-phase separation for renaturation of inclusion bodies (14). In the present study, for the improvement of refolding process, we have selected dilution and dialysis methods. In on-column purification, we